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Advances in Brief
Dependence of Histone Modifications and Gene Expression on DNA Hypermethylation in Cancer
Jill A. Fahrner, Sayaka Eguchi, James G. Herman and Stephen B. Baylin
Jill A. Fahrner
Sayaka Eguchi
James G. Herman
Stephen B. Baylin
DOI: Published December 2002
We examined the relationship between aberrant DNA hypermethylation and key histone code components at a hypermethylated, silenced tumor suppressor gene promoter in human cancer. In lower eukaryotes, methylated H3-lysine 9 (methyl-H3-K9) determines DNA methylation and correlates with repressed gene transcription. Here we show that a zone of deacetylated histone H3 plus methyl-H3-K9 surrounds a hypermethylated, silenced hMLH1 promoter, which, when unmethylated and active, is embedded in methyl-H3-K4 and acetylated H3. Inhibiting DNA methyltransferases, but not histone deacetylases, leads first to promoter demethylation, second to gene reexpression, and finally to complete histone code reversal. Our findings suggest a new paradigm-DNA methylation may directly, or indirectly by inhibiting transcription, maintain key repressive elements of the histone code at a hypermethylated gene promoter in cancer.
Aberrant promoter DNA hypermethylation and associated epigenetic gene silencing frequently provide for loss of tumor suppressor gene function in cancer (1 , 2) . DNA methylation-mediated gene silencing is closely linked to the deacetylation of histones (1 , 2) . More recently, methylation of histones at key lysine residues has been shown to work in concert with acetylation and other modifications to provide a histone code that may determine heritable transcriptional states (3) . The data for this have come predominantly from studies examining broad domains in chickens (4) , yeast (5) , and the mammalian inactive X chromosome (6, 7, 8) , as well as individual promoter sites known to be expressed or silenced by epigenetic mechanisms such as X inactivation (8) and genomic imprinting (9) . These studies reveal that acetylated histone H3 and methyl-H3-K45 are enriched in euchromatic domains and correlate with active gene expression, whereas methyl-H3-K9 is enriched at deacetylated, transcriptionally silent heterochromatic regions (4, 5, 6, 7, 8, 9) .
The above histone modifications have been widely hypothesized to determine active versus inactive gene expression status (4, 5, 6, 7, 8, 9) . Moreover, presence of methyl-H3-K9 has recently been shown to be essential for all or a subset of DNA methylation in Neurospora crassa (10) and Arabidopsis thaliana (11) , respectively. However, for hypermethylated tumor suppressor genes in human cancer, DNA hypermethylation appears to be dominant over at least the histone deacetylation part of the histone code for maintaining a silenced state (12) . In this regard, we have shown previously that the DNA demethylating agent 5-Aza-dC, but not the HDAC inhibitor TSA, reactivates the expression of such genes (12 , 13) . We now provide evidence that promoter DNA hypermethylation can control transcriptional silencing and, either directly or indirectly, the state of key elements of the histone code. At hMLH1, a mismatch repair gene often silenced with aberrant CpG island hypermethylation in colorectal cancers (14) , a zone of deacetylated H3 (deacetylated histone H3-K9 and -K14) plus methyl-H3-K9 (dimethyl-H3-K9) surrounds the hypermethylated, silenced promoter. This same promoter, when unmethylated and active, is embedded in methyl-H3-K4 (dimethyl-H3-K4) and acetylated H3 (acetylated histone H3-K9 and -K14). Treatment with TSA fails to reactivate the hypermethylated gene or dramatically alters the histone modifications examined. However, 5-Aza-dC treatment leads to initiation of demethylation by 12 h, appearance of transcription by 24 h, and full reversal of key elements of the histone code by 48 h. Thus, DNA hypermethylation, either directly or indirectly through suppressing transcription, appears to specify for repressive histone modifications at a tumor suppressor gene promoter. An important element of the findings is that the demethylating drug 5-Aza-dC appears to be a potent tool for dissecting the components of this DNA methylation-mediated transcriptional control and for potentially reversing their interaction for therapeutic purposes in cancer.
Cell Culture.
SW480 cells were maintained in McCoy's 5A modified medium. RKO cells were maintained in MEM. All media (Invitrogen) were supplemented with 10% fetal bovine serum (Gemini Bio-Products) and 1% penicillin/streptomycin (Invitrogen) and grown at 37°C in 5% CO2 atmosphere.
5-Aza-dC and TSA Treatments.
Cells were treated with mock or 1 μm 5-Aza-dC (Sigma) for 12, 24, 48 h, or 5 days or with 300 nm TSA (Wako) for 24 h, as described previously (12 , 14) .
ChIP.
We used the ChIP Assay Kit from Upstate Biotechnology and followed the manufacturer's protocol with some modifications.
Briefly, proteins were cross-linked to DNA by addition of formaldehyde directly to the culture medium to a final concentration of 1% for 10 min at room temperature. The cross-linking reaction was quenched by adding glycine to a final concentration of 0.125 m for 5 min at room temperature. The medium was then removed and cells were washed with 1× PBS containing a combination of protease inhibitors (1 mm Pefabloc and 1× Complete protease inhibitor mixture; Roche Molecular Biochemicals). The PBS was removed and 0.2× trypsin was added to the cells. After a 5-min incubation at 37°C, ice-cold 1× PBS containing 10% FBS was added to stop trypsinization. The cells were scraped off the culture flask, pelleted, and washed twice with 1× PBS plus protease inhibitors as above. For each ChIP assay ∼106 cells were used. The sonicated samples were precleared with 80 μl of salmon sperm DNA/Protein A and Protein G agarose beads (3:1 ratio of Protein A to Protein G; Upstate Biotechnology) for 1 h at 4°C with agitation. The soluble chromatin fraction was collected, and 5 μl of either anti-acetyl-Histone H3 (Lys 9 and Lys 14), anti-dimethyl-Histone H3 (Lys 4), anti-dimethyl-Histone H3 (Lys 9), or no antibody was added and incubated overnight with rotation (all antibodies from Upstate Biotechnology). Immune complexes were collected with 60 μl of the 3:1 salmon sperm DNA/Protein A and Protein G agarose beads. The beads were washed as recommended but were transferred to a new tube before each wash. After elution, the cross-links were reversed, and the samples were digested with proteinase K. DNA was recovered by phenol extraction, ethanol precipitated, and resuspended in 1× 10 mm Tris (pH 8)-1 mm EDTA buffer.
PCR Amplification and Analysis.
Primer sets for PCR were designed to amplify overlapping fragments of ∼200 bp along the hMLH1 promoter. One primer set for GAPDH was designed to amplify a 128-bp fragment of the genomic sequence to serve as an internal control. All primers were purchased from Invitrogen or IDT. All PCR reactions were performed with JumpStart REDTaq DNA Polymerase (Sigma) in a total volume of 25 μl, using 1–2 μl of either immunoprecipitated (bound) DNA, a 1:10 dilution of nonimmunoprecipitated (input) DNA, or a no-antibody control. All reactions were optimized with input DNA to ensure that PCR products for both hMLH1 and GAPDH were in the linear range of amplification. Primer sequences and additional PCR conditions are available upon request. Ten μl of PCR product were size fractionated by PAGE and were quantified using Kodak Digital Science 1D Image Analysis software. Enrichment was calculated by taking the ratio between the net intensity of the hMLH1 PCR product from each primer set and the net intensity of the GAPDH PCR product for the bound sample and dividing this by the same ratio calculated for the input sample. Values for enrichment were calculated as the average from at least two independent ChIP experiments and multiple independent PCR analyses of each.
MSP.
Genomic DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega). The genomic DNA was modified by bisulfite treatment, as described previously (15) . The primers used for MSP have been previously described (14) and were purchased from Invitrogen. Primer sequences and additional PCR conditions are available upon request.
Reverse Transcriptase-PCR.
We isolated RNA with Trizol (Invitrogen), according to the manufacturer's instructions. RNA was reverse transcribed using Superscript II Rnase H Reverse Transcriptase (Invitrogen). PCR was performed using 1 μl of cDNA and primers unambiguous for GAPDH or hMLH1 (Invitrogen). All PCR reactions were performed with JumpStart REDTaq DNA Polymerase (Sigma) in a total volume of 25 μl. Primer sequences and additional PCR conditions are available upon request.
Mapping the Histone Code at hMLH1.
We compiled a detailed map of histone acetylation and histone methylation across a 1.8-kb region of the promoter for hMLH1. We performed ChIP for two human colorectal cancer cell lines, RKO and SW480, in which the hMLH1 promoter is hypermethylated and transcriptionally silenced or unmethylated and transcriptionally active, respectively (14) . The histone-associated DNA regions were analyzed using a multiplex PCR approach with overlapping primer sets spanning the promoter (Fig. 1A)⇓ . Overall, acetylated histone H3 was enriched throughout the unmethylated hMLH1 promoter; however, there was essentially no acetylation of these same sites along the hypermethylated promoter (Fig. 1, B and C)⇓ . Virtually identical results were observed for methyl-H3-K4 at the two promoters (Fig. 1, D and E)⇓ . In stark contrast, methyl-H3-K9 was enriched along the entire hypermethylated, silenced hMLH1 promoter, especially over a region where it was depleted to virtually undetectable levels along the unmethylated, transcriptionally active promoter (Fig. 1, F and G)⇓ . Thus, it seems that key elements of the histone code surrounding an aberrantly hypermethylated tumor suppressor gene in human cancer resemble the histone modifications that may be necessary for proper genomic imprinting and gene expression along the X chromosome in other mammalian cells (6, 7, 8, 9) and for defining large heterochromatic and euchromatic domains in chickens (4) and yeast (5) .
Map of histone H3 modifications along a hypermethylated versus an unmethylated hMLH1 promoter. A, schematic of the hMLH1 promoter. The vertical lines represent the location of CpG dinucleotides, and the arrow indicates the approximate position of the transcription start site. The CpG island extends 3′ from ∼−800 (relative to the transcription start site) into exon 1. The doubled horizontal line denotes the region examined by MSP. In SW480 cells, the promoter is unmethylated, and the gene is expressed. However in RKO cells, the promoter is hypermethylated, and the gene is silenced. The horizontal bars below the schematic indicate the location of the DNA fragments amplified by PCR done on the DNA recovered from ChIP experiments. The broken bars denote the primer sets used in the time course experiments. B, D, and F, enrichment of hMLH1 promoter DNA immunoprecipitated with antibodies specific for acetylated histone H3 (K9 and K14), dimethyl-H3-K4, and dimethyl-H3-K9, respectively. Points on the graphs represent data from the corresponding DNA fragment amplified by PCR, as shown at the bottom A. The value of each point was calculated as the average from two independent ChIP experiments and a total of four independent PCR analyses. Each error bar indicates the SD from the mean. □ represent data from SW480. ▪ represent data from RKO. C, E, and G, representative PCR analyses of ChIP on RKO and SW480 from areas typical of enrichment for acetylated H3, methyl-H3-K4, and methyl-H3-K9, respectively. Multiplex PCR was performed on bound (B) immunoprecipitated DNA and input (I) nonimmunoprecipitated DNA with each hMLH1 primer set.
Inhibition of HDACs with TSA.
Although little is known about the relationship between DNA methylation and the methylation component of the histone code in mammals, it has been known for some time that DNA methylation and histone deacetylation work in concert to silence genes in cancer (1 , 2 , 16 , 17) . In fact, we have shown previously that DNA methylation is dominant over histone deacetylation in maintaining a silent state at hypermethylated promoters because 5-Aza-dC can reactivate genes silenced with aberrant promoter hypermethylation but TSA alone cannot reactivate these same genes (12) . We explored these findings further at the level of the histone code. We treated RKO cells with TSA, which alone did not reactivate hMLH1, but which was able to reactivate FABP4, a control gene silenced in these cells by a mechanism distinct from one involving DNA hypermethylation (Ref. 13 ; data not shown). Subsequent ChIP and PCR analysis revealed only a slight increase in acetylated H3 at the hMLH1 promoter (Fig. 2, A and B)⇓ and essentially no change in methyl-H3-K4 (Fig. 2, C and D)⇓ and methyl-H3-K9 (Fig. 2, E and F)⇓ . These data show that in addition to being unable to reactivate expression of a hypermethylated, silenced hMLH1gene, TSA alone is unable to evoke obvious changes in key parameters of the histone code at this promoter.
Inhibition of histone deacetylation by TSA fails to dramatically alter key components of the histone code map along the hypermethylated hMLH1 promoter. ChIP was done on RKO cells after treatment with 300 nm TSA for 24 h. A, C, and E, enrichment of acetylated histone H3 (K9 and K14), dimethyl-H3-K4, and dimethyl-H3-K9, respectively, at the hMLH1 promoter. ○ represents enrichment in RKO cells treated with TSA. • represents data from untreated RKO cells. Points on each graph represent data from the corresponding DNA fragment amplified by PCR, as illustrated in Fig. 1A⇓ . The value of each point was calculated as the average from two independent ChIP experiments and a total of four independent PCR analyses. Each error bar indicates the SD from the mean. B, D, and F, representative PCR analyses of ChIP performed on RKO cells, before and after treatment with TSA, from areas typical of enrichment for acetylated H3, methyl-H3-K4, and methyl-H3-K9, respectively. Bound DNA (B) and input DNA (I) were coamplified with primers for hMLH1 and GAPDH.
Inhibition of DNMTs with 5-Aza-dC.
Recent genetic studies in Arabidopsis (11) and Neurospora (10) suggest that methyl-H3-K9 may determine sites of DNA methylation, although evidence for the existence of this relationship in higher eukaryotes has not been explored. We therefore set out to probe this relationship between key elements of the histone code and DNA methylation. We addressed this question by using ChIP and PCR analysis to examine the fate of histone modifications at the hMLH1 promoter upon inhibition of DNMTs by a dose of 5-Aza-dC, which is sufficient to cause demethylation of the promoter region (14) and to reactivate the expression of the hypermethylated, silenced hMLH1 gene in RKO cells (Ref. 14 ; data not shown). Surprisingly, after drug treatment, we observed a complete reversal of the histone code components examined at the hMLH1 promoter in RKO cells. Acetylated H3 and methyl-H3-K4 levels became markedly enriched (Fig. 3, A–D)⇓ , whereas methyl-H3-K9 levels were severely depleted (Fig. 3, E and F)⇓ . Thus with 5-Aza-dC, we recapitulated in RKO cells the state of the unmethylated, expressed promoter originally observed in SW480 cells (compare Fig. 3, A, C, and E⇓ to Fig. 1, B, D, and F⇓ ). This transformation of key parameters of the histone code upon inhibition of the DNMTs suggests that in human colorectal cancer cells, DNA hypermethylation, or another activity mediated by DNMTs, may be essential for maintaining a particular combination of histone modifications at gene promoters silenced with aberrant DNA hypermethylation. Furthermore, the observation that 5-Aza-dC, but not TSA, can both reactivate expression of the silenced hMLH1 gene and completely reverse key histone modifications surrounding the gene promoter strengthens the idea that there exists some interdependence between reversal of important histone code components and reactivation of a gene silenced with aberrant DNA hypermethylation.
Inhibition of DNA methylation by 5-Aza-dC completely reverses all examined components of the histone code map along the hypermethylated hMLH1 promoter. ChIP was performed on RKO cells after treatment with 1 μm 5-Aza-dC for 5 days. A, C, and E, enrichment of hMLH1 promoter DNA precipitated by antibodies specific for acetylated histone H3 (K9 and K14), dimethyl-H3-K4, and dimethyl-H3-K9, respectively. ○ represents enrichment in RKO cells treated with 5-Aza-dC. • represents data from untreated RKO cells. The value of each point was calculated as the average from two (untreated) or three (drug-treated) independent ChIP experiments and four independent PCR analyses from each untreated or drug-treated experiment. Each error bar indicates the SD from the mean. Points on each graph correspond to the overlapping DNA fragments amplified by PCR as depicted in Fig. 1A. B, D, and F⇓ , representative PCR analyses of ChIP done on RKO cells, with or without treatment with 5-Aza-dC, from areas typical of enrichment for acetylated H3, methyl-H3-K4, and methyl-H3-K9, respectively. DNA from bound (B) and input (I) fractions were coamplified with primers for hMLH1 and GAPDH.
Time Course Analysis after 5-Aza-dC Treatment.
The observation that inhibition of the DNMTs leads to both steady-state reactivation of hMLH1 expression and complete reversal of key histone code parameters surrounding the gene promoter invited delineating the sequence of events to help dissect the operative mechanisms. We performed time course studies in which RKO cells were treated with 5-Aza-dC and monitored over 5 days for the states of key elements of the histone code, DNA methylation, and gene expression using ChIP and PCR analysis, MSP, and reverse transcriptase-PCR, respectively. For the ChIP and PCR analysis, we used four of the original 13 primer sets (Fig. 1A)⇓ , which cover the region of greatest difference in histone modification observed between RKO and SW480 cells and also between 5-Aza-dC-treated and -untreated RKO cells at hMLH1 (Fig. 1, A, B, D, and F⇓ ; Fig. 3, A, C, and E⇓ ). At 12 and 24 h after the start of 5-Aza-dC treatment, there was no dramatic change in the histone code components examined (Fig. 4)⇓ . By 48 h, acetylated H3 and methyl-H3-K4 showed dramatic enrichment in 5-Aza-dC-treated samples compared with mock-treated samples; at the same time, methyl-H3-K9 became depleted (Fig. 4)⇓ . We next used MSP to examine the methylation status of the hMLH1 promoter after treatment with 5-Aza-dC. The region examined covers the area of greatest CpG density in the promoter and overlaps with the region examined by ChIP and PCR analysis in these time course studies (Fig. 1A)⇓ . By 12 h, we observed demethylation of the promoter, which was maximal by 24 h and sustained until 5 days after the start of drug treatment (Fig. 5A)⇓ . Finally, we examined reexpression of hMLH1 by 5-Aza-dC using reverse transcriptase-PCR. Transcriptional reactivation became apparent by 24 h after the start of 5-Aza-dC treatment, and gene expression continued throughout the time course (Fig. 5B)⇓ . The observed sequence of events, then, is demethylation of the hMLH1 promoter by 12 h, appearance of hMLH1 transcript by 24 h, and complete reversal of all examined histone code components along the gene promoter by 48 h (Table 1)⇓ .
Treatment with 5-Aza-dC reverses all components of the histone code examined at a hypermethylated hMLH1 promoter by 48 h. The data represent two independent time course experiments in which RKO cells were treated with 1 μm 5-Aza-dC (or mock-treated) and harvested at each time point shown for ChIP analysis. A total of four to seven PCR analyses were performed on the immunoprecipitated DNA from each time point. Each point on the graphs represents the average value of enrichment, and each error bar indicates the SD from the mean. ○ represents data from RKO cells treated with 5-Aza-dC. • represents data from mock-treated RKO cells. Data from a 5-day time point served as positive controls to ensure that drug treatment was effective. The broken horizontal bars under the hMLH1 promoter schematic in Fig. 1A⇓ indicate the location of the primer sets used in this ChIP and PCR analysis.
Treatment with 5-Aza-dC initiates demethylation by 12 h and transcription by 24 h at a hypermethylated hMLH1 promoter. RKO cells were treated with 1 μm 5-Aza-dC and harvested at the indicated time points. A, MSP analysis of hMLH1. A doubled line above the hMLH1 promoter schematic in Fig. 1A⇓ indicates the region examined by MSP. Methylation was detected by the presence of a PCR product amplified by methylation-specific primers in the M lanes. Demethylation was detected by PCR products amplified by unmethylated-specific primers in the U lanes. Bisulfite dH2O denotes bisulfite-treated dH2O, which served as a negative control for the treatment. RKO and SW480 served as positive controls for the methylated and unmethylated PCR reactions, respectively. B, reverse transcriptase-PCR analysis of hMLH1 expression. GAPDH expression served as a loading control. Five-day mock- and 5-day 5-Aza-dC-treated RKO cells served as negative and positive controls, respectively, for hMLH1 expression.
Summary of time course data
Although in these experiments it appears that demethylation distinctly occurred first, as it was detectable by 12 h and maximal by 24 h, we are less certain about the order of events with respect to reactivation of transcription and reversal of the histone code parameters examined because of the different sensitivities of the techniques used. To help sort this out, we examined data from several immunofluorescence experiments in which we stained for hMLH1 protein after treatment of RKO cells for 24 h with the same dose of 5-Aza-dC used in the present studies. No nuclear staining was visible in mock-treated cells, but distinct reexpression of hMLH1 protein was present in 33–50% of RKO cells by 24 h (data not shown). These data suggest that a substantial percentage of cells were transcribing hMLH1 by 24 h in our present time course experiments and that the ChIP procedures would likely have detected a distinct change in the histone code parameters examined if these changes had preceded transcription. Our data, then, suggest a sequence of events in which 5-Aza-dC produces demethylation first, transcriptional reactivation second, and reversal of important histone code components third.
Our data provide the first detailed map of H3 acetylation and H3 methylation for a hypermethylated versus an unmethylated gene promoter in cancer cells. We also demonstrate here that inhibiting the DNMTs, but not the HDACs, essentially recapitulates at a hypermethylated, silenced promoter a combination of histone modifications similar to that at an unmethylated, active promoter. Finally, our results show unequivocally that DNA demethylation precedes both the reactivation of the silenced gene and, somewhat surprisingly, the reversal of key elements of the repressive histone code. These findings are consistent with the idea that DNA demethylation, either directly or indirectly by reactivating transcription of the hMLH1 gene, reverses important components of the repressive histone code surrounding the hypermethylated promoter. These results favor the idea that DNA hypermethylation, not a particular combination of histone modifications containing elevated methyl-H3-K9, is the dominant epigenetic mechanism involved in maintaining silencing of the hMLH1 gene.
In considering the mechanisms that underlie our observation that upon treatment with 5-Aza-dC, demethylation precedes reactivation of transcription, which precedes reversal of key histone code parameters, at least two scenarios may be considered. The first potential mechanism is one in which DNA methylation plays a direct role in both gene silencing and maintaining a repressive histone code at a hypermethylated gene promoter in cancer. We could speculate that the DNA modification itself, or components of the DNA methylating machinery such as the DNMTs or methyl-CpG binding proteins, could directly interact with histone methyltransferases or proteins that target them, directing them to regions containing DNA methylation and allowing them to set up a repressive histone code (18) . If this turns out to be the case, it would suggest a new paradigm, seeing that data from Neurospora (10) and Arabidopsis (11) suggest the opposite and point to a role for methyl-H3-K9 in targeting and maintaining DNA methylation. Our data stress the importance of identifying the enzymes responsible for modifying the histones in the setting of mammalian gene promoters and developing histone methyltransferase inhibitors to formally test relationships between histone modifications and DNA methylation in mammalian cells.
A second and more indirect mechanism may better fit the changes we have observed and relate to an important new view of relationships between histone code parameters and gene transcription (19, 20, 21) . In this scenario, DNA demethylation leads to gene reactivation, which in turn, leads to reversal of key elements of the histone code. This possibility is supported by our temporal data and by recent exciting findings in Arabidopsis (21) and Drosophila (20) by others. Johnson et al. (21) report that loss of DNA methylation itself does not lead to a decrease in methyl-H3-K9; rather, only at loci where reactivation of transcription occurs because of loss of DNA methylation does methyl-H3-K9 decrease. They postulate that methyl-H3-K9 may be replaced by replication-independent deposition of new nucleosomes containing variant histone H3.3 once transcription occurs (21) , a concept suggested by studies from Ahmad and Henikoff (20) in Drosophila. In light of these findings, our data could be interpreted as showing that 5-Aza-dC leads to demethylation of the DNA, which causes reactivation of hMLH1 gene transcription and, possibly, subsequent deposition of H3.3. The newly deposited variant histones would lack methyl-H3-K9 and could undergo posttranslational modification, including methylation at K4 or acetylation, resulting in a heritable histone code that supports active transcription at the hMLH1 promoter. This type of mechanism could also help to explain our previous findings that TSA alone cannot reactivate hypermethylated genes in cancer but can synergize with low doses of 5-Aza-dC to reactivate such genes (12 , 13) . In this model, TSA may be working by facilitating the acetylation of the newly deposited histones, thus helping to augment newly initiated transcription.
Although additional studies must continue to verify the above proposed sequence of events, our new findings are important to multiple aspects of abnormal, epigenetically mediated gene silencing in cancer. Pooling all of the available data, including ours and those from studies in Neurospora (10) and Arabidopsis (11 , 21) , the following sequence of events is a plausible model for DNA methylation-mediated silencing of tumor suppressor genes in cancer. Our extensive histone code map along the hMLH1 promoter in SW480 cells suggests that enrichment of acetylated H3 and methyl-H3-K4 within and upstream of promoter CpG islands could protect the islands at normally expressed mammalian genes from DNA hypermethylation, similar to the postulated methyl-H3-K4- and acetylation-mediated protection from transcriptional repression that has been suggested to occur in chickens (4) and yeast (5) . Such protection may be lost in some cancers at selected sites because these key components of the histone code break down, allowing histone deacetylation to occur, methyl-H3-K9 to spread into the promoter, and aberrant DNA hypermethylation of the CpG island and silencing to result. Our data suggest that DNA hypermethylation firmly maintains this new heritable silenced state by repressing transcription and, directly or indirectly, sustaining these key elements of a repressive histone code. Importantly, 5-Aza-dC is able to disrupt this established heritable state of the histones. These findings stress the usefulness of this drug for dissecting the basic relationships between DNA methylation and histone modifications for their contribution to gene expression patterns in normal and disease states, as well as the possibilities for reversing DNA hypermethylation and repressive components of the histone code for prevention and treatment of cancer.
We thank Drs. Kurtis E. Bachman and Kam-Wing Jair for helpful suggestions during the course of these studies. We also thank Dr. Elizabeth E. Cameron for immunofluorescence data mentioned in the text.
↵1 Supported by NIH Grant CA43318 from the National Cancer Institute and ES11858 from the National Institute of Environmental Health Sciences.
↵2 J. G. H. and S. B. B. are consultants to Tibotec-Virco. Under licensing agreement between the Johns Hopkins University and Tibotec-Virco, MSP was licensed to Tibotec-Virco, and they are entitled to a share of the royalties received by the University from sales of the licensed technology. The terms of these arrangements are being managed by the University in accordance with its conflict of interest policies.
↵3 These authors contributed equally to this work.
↵4 To whom requests for reprints should be addressed, at The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Tumor Biology Laboratory, The Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, Room 541, Baltimore, MD 21231-1000. Phone: (410) 955-8506; Fax: (410) 614-9884; E-mail: sbaylin{at}jhmi.edu
↵5 The abbreviations used are: methyl-H3-K4, methylated histone H3-lysine 4; methyl-H3-K9, methylated histone H3-lysine 9; DNMT, DNA methyltransferase; HDAC, histone deacetylase; 5-Aza-dC, 5-Aza-2′deoxycytidine; TSA, Trichostatin A; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MSP, methylation-specific PCR.
Received November 11, 2002.
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Johnson L., Cao X., Jacobsen S. Interplay between two epigenetic marks: DNA methylation and histone H3 lysine 9 methylation. Curr. Biol., 12: 1360-1367, 2002.
You are going to email the following Dependence of Histone Modifications and Gene Expression on DNA Hypermethylation in Cancer
Cancer Res December 15 2002 (62) (24) 7213-7218;
Crucial Role of Phospholipase Cε in Chemical Carcinogen-Induced Skin Tumor Development
Down-Regulation of Regulatory Subunit Type 1A of Protein Kinase A Leads to Endocrine and Other Tumors
Activating Mutations of the Noonan Syndrome-Associated SHP2/PTPN11 Gene in Human Solid Tumors and Adult Acute Myelogenous Leukemia
Show more Advances in Brief


Finding the best ways to stay in touch via messaging, social networking, phone calls, and more.
Snappables use augmented reality technology in tandem with the front-facing camera and can be controlled with touch, motion, and even facial expressions.
The Data Download feature will allow anyone to obtain a file with their photos, videos, messages, and additional information from the social network.
In June, Twitter is making major changes that will break push notifications and automatic timeline refresh on third-party apps.
Arriving in beta with iOS 11.3, Business Chat allows iPhone or iPad users to ask questions and more from a number of companies.
If approved, the 13 different accessibility emojis - like a service dog with a vest - could arrive in iOS devices sometime in 2019 with Emoji 12.0.
A great third-party keyboard option, SwiftKey will soon be adding a new Toolbar option that will provide quick access to emojis, GIFs, and more.
The new feature for the Amazon Alexa app on the iPad should allow even more people to use the ecosystem and take advantage of the communication features.
Along with the ability to search directly from iMessage, the Google app update also brings drag and drop support for the iPad.
A long-requested addition, the new Bookmarks feature will allow Twitter app users to save all types of content for later viewing.
Swype keyboards are no more as Nuance moves away from the consumer market. The popular third-party keyboard was also pulled for Android devices.
Some of the 157 new emoji landing on the iPhone and iPad later this year include a superhero, supervillain, skateboarding, and more.
A nasty Facebook Messenger bug has been uncovered that's affecting some iOS users. The bug affects typing inside the app.
The once hugely-popular communication program AOL Instant Messenger has shut down as of today after 20 years in service.
After officially doubling the maximum character count per tweet, Twitter is giving users another feature to help voice their opinion with Threads.
A new Instagram feature will allow users to follow specific hashtags and see top posts in your feed and see Stories as well.
The direct messaging app, called Direct, is available in six countries. It removes the functionality from the main Instagram app.
The new Stories Archive feature will allow users to revisit their stories after they expire. With Stories Highlights, you can create best-of moments.
Reversible emoji could be in our future. If approved, Apple and other vendors would be able to choose which emoji can be reversible on their devices.
Facebook Messenger Kids is a new app launching this week for iOS. Don't worry, parents, it's not nearly as scary as it sounds.
Instagram is apparently busy testing a number of new features in its popular photo-sharing app that may appear for everyone soon.
The new Snapchat filters can recognize a number of different objects including concerts, beaches, pets, sports, and food.
Less than 24 hours after Twitter announced the new tweet character limit, a new Tweetbot update has arrived to support the major change.
The team behind the app says Alto Mail is discontinued, so you'll need to find a new email app. Jeff Byrnes has the news and some options for you.


Welsh Women's Aid is looking to expand our pool of associates to provide independent assessment of our national quality service standards; to help deliver our capacity building project that supports specialist services across Wales to evidence their impact and become commission-ready; and to deliver domestic abuse/violence against women training in a range of subject areas.
We are keen to receive expressions an interest from consultants skilled and experienced domestic abuse/violence against women experts with a proven track record of supporting small specialist refuge and community services, of delivering training programmes, and providing a range of bespoke consultancy work related to strategic and operational responses to domestic abuse.
Please click here to download further information.


Love the name...right up my alley, so I had to try it! Lol Delicious coffee!
I love that it's sustainably farmed, fair trade and organic, plus the price is great. It's delicious too! It's my new go-to coffee.
Really love the flavor of this coffee. It's a bit expensive, but I wanted to splurge to try it out. Glad I did.
I like this lighter roast coffee. I would order again.
Will be ordering this regularly!


Made exclusive for i-D, it's all you need for a good time.
JD. Reid is the lowkey brilliant North London producer whose powerful new Calibrate EP, out now on Rinse, featured Kojey Radical, 808INK, Oscar Worldpeace, Hodgy from Odd Future, and Novelist. A strong line up if we ever saw one. The release emerged back in July, and followed up his D Double E featuring 2016 debut io EP, as well as production work with various artists including Mabel and what is arguably her greatest single, Finders Keepers.
To celebrate the end of a summer that had its ups and downs but was ultimately not bad in terms of soundtracks, JD has made us a bassy mix that goes hard and fast. With a heavy and very welcome dose of his own works (that Kendrick rework from his EDITS + BEATS VOL.2 release rules), the mix is as eclectic as the selector, making Ivy Lab, Dizzee, Gaslamp Killer, Rox FOE, and Lil Uzi Vert seem like one big happy family.
"When I started the project, there was not a clear plan," JD has said of his recent EP, "but the goal was to make sense of something abstract - and that, by definition, is to calibrate." Sounds to us like he's gone and done it again. Happy Friday!


Want to buy properties already rented and have a good history of rentals for steady income and ROI? Look no more. Frequently throughout the year, we at Phoenix Realty & Property Management have owners/investors who are looking to exit their RE investments. Reasons are many but the top three are retirement, 1031 exchange, and/or they have achieved their ROI based on depreciation schedule.
List below are tenant occupied homes for sale in Durham, Raleigh and surrounded areas. The leases are active so the new owners will need to agree to live out thru the leases.
Central Air Coolinig, Forced Air Heating, Attic, Cable Ready, Ceiling Fan, Large Sun Room, single Garage with Front and Back Access, Hardwood and Tile Flooring, Nice Private Front and Back Yard and Lawn.
Beautiful one story ranch in the historical Rockwood neighborhood within three miles of Duke University. It is within walking distance (literally) of Nana Restaurant, Nanataco, The Original QShack, The Boot, and more. Home is seated deep into the lot not easily view from University Drive giving much privacy. Surrounding neighbors are mid-century homes at much higher prices. Currently, the home is leased going into its second year. Contact Phoenix Realty to arrange a showing. The property is not listed in the MLS per the owner's request. This property is perfect for an investor wanting to acquire a value home with much upside potential and with an immediate cash flow. It has a very nice private backyard with thick bushes and trees buffering the Property. Please see the pictures provided. The home has many updates and a newer HVAC system. You must respect the tenant's current lease terms and privacy. Please do not try to access the home on your own; you must have an appointment.
Tucked behind Colony Lake community without HOA fees. Easy and short access to 15-501 HWY within easy reach to UNC campus and Duke. Simple open floor plan with updated look and feel. Incredible privacy with a fenced back yard. Extra large deck to enjoy the privacy. Lot has two open gravel parks. Currently tenant is living there to mid June 2018. You must respect and uphold the active lease.
* The 1031 exchange can be a powerful tool for investors. Under IRC Section 1031 (a) (1) IRS does not recognize capital gains or losses when an investor exchanges a property used for conducting business for a like-kind property that will be used for business or for investment purposes. While the exchange is tax-deferred, it's still a taxable event, so using a 1031 exchange must be done properly in order to take advantage of the deferral options.


text:
Q: Sequelize - multi-condition queries I'm using Sequelize to query a PostGIS database and return user records based on geographic proximity. I want to exclude the current user from the search results, but I can't figure out the proper syntax to add multiple conditions to this particular kind of search.
As it stands, the query is
models.Geometry.findAll({
        where: models.sequelize.where(
            models.sequelize.fn(
                'ST_Distance_Sphere',
                models.sequelize.col('the_geom'),
                models.sequelize.fn(
                    'ST_GEOMFROMTEXT',
                    'POINT(' + row.dataValues.longitude + ' ' + row.dataValues.latitude + ')', 4326
                )
            ),
            '<=',
            radius
        )
    })

I'd like to add
where: {
     UserId: {
         $not: currentUser.id
     }
}

I couldn't find anything in the Sequelize docs with an example... the closest I got was
Post.findAll({
     where: sequelize.where(sequelize.fn('char_length', sequelize.col('status')), 6)
});

but this is not really any different than my current situation.

A: You can assign the models.sequelize.where function value to some attribute in the where object - this attribute will not be used as a lookup field
models.Geometry.findAll({
    where: {
        stDistanceSphere: models.sequelize.where(
            models.sequelize.fn(
                'ST_Distance_Sphere',
                models.sequelize.col('the_geom'),
                models.sequelize.fn(
                    'ST_GEOMFROMTEXT',
                    'POINT(' + row.dataValues.longitude + ' ' + row.dataValues.latitude + ')', 4326
                )
            ),
            '<=',
            radius
        ),
        UserId: { $not: currentUser.id }
    }
})

Or you can use the sequelize.and() function to construct AND query
models.Geometry.findAll({
    where: models.sequelize.and(
        models.sequelize.where(
            models.sequelize.fn(
                'ST_Distance_Sphere',
                models.sequelize.col('the_geom'),
                models.sequelize.fn(
                    'ST_GEOMFROMTEXT',
                    'POINT(' + row.dataValues.longitude + ' ' + row.dataValues.latitude + ')', 4326
                )
            ),
            '<=',
            radius
        ),
        { UserId: { $not: currentUser.id } }
    )
})

redpajama_set_name: RedPajamaStackExchange


« Bark at the Park: Take Your Dogs to Watch the Seattle Mariners!
Spring on in for some spring-tastic fun at our "Paws"-itively Springy Photo Booth! Photos are compliments of Mud Bay and will be emailed the week after the event.


Ever heard of the Mai Tai? If you have then you know how great it is, if you haven't then you're doing life all wrong. Funnily enough it originated at Trader Vics on Park Lane. I genuinely had no idea - I guess I expected it to originate from some Caribbean island or something.
The Mai Tai is also synonymous with Trader Vic's as it was created in 1944 in Oakland, California by Victor J. Bergeron, also known as Trader Vic. This world famous cocktail uses dark and light rum, fresh lime, orange curaçao, a dash of rock candy syrup and a drop of French Orgeat. On serving it to friends from Tahiti, they exclaimed "Mai ta roa ae", which translates as 'out of this world, the best' – and so, the Mai Tai was born.
We arrived and immediately are presented with Mai Tais...this is my kind of Thursday. It was genuinely the best I've ever had; fruity, smooth and tasted far too good thus resulting in drinking far too many.
Trader Vics currently are running Lei'd Back Sessions with a host of singers providing entertainment for all. This particular evening the soulful songstress Loretta Heywood, best known for her ethereal vocals with 90's electronic band Bomb The Bass, was there ready to provide us all with incredible music.
Before Loretta Heywood took the stage - we enjoyed some cocktails and food. The bartender, who was lovely, can pick a cocktail depending on what flavours you liked. Personally preferring rum based cocktails with a sweeter taste rather than anything else he, of course, recommended the a Mango based smoothie which tasted much like the smoothies I make in the morning - either he's doing something right, or I'm doing something wrong.
The atmosphere at the Lei'd Back Sessions on this particular Thursday was buzzing - there was a good amount of people without it being too busy and crammed. Not to be ironic - but it really was 'lei'd back' and that suited me down the ground. You can enjoy a cocktail (or, in our case about 7) whilst having a natter and enjoying the music.
We also enjoyed some food (created in their wood-fired ovens using natural smoke and heat) in which we had various nibbles from pork in a tangy sauce, which we cooked over a lit flame, to deep-fried calamari. It was good. After a couple of heavy meals the previous nights the light snacks were more than welcomed.
The performance began around 8.30pm - but it's a Thursday and as I always say Thursday is the new Friday. Loretta genuinely lit up the room with her voice the moment she begun her performance - I think I speak for the whole room when I say we were immediately captivated by her. I could have listened to her for hours.
Now I am only giving you a snippet into what Lei'd Back Sessions at Trader Vics are like - and believe me I'd really recommend heading there yourselves. The third gig is on 5th May with sultry Blues singer Annie Bea, who has what some call an encyclopaedic knowledge of jazz lyrics influencing her tone and style. Tickets are £10 and include a cocktail.


What Is The Highest Grossing Christmas Film Of All Time?
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text:
és una jugadora d'hoquei sobre gel finlandesa, ja retirada, que va competir entre el 1983 i el 2000. Jugava de davantera.

El 1998 va prendre part en els Jocs Olímpics d'Hivern de Nagano, on guanyà la medalla de bronze en la competició d'hoquei sobre gel.

En el seu palmarès també destaquen cinc medalles de bronze al Campionat del món i tres medalles d'or al Campionat d'Europa. Amb la selecció finlandesa jugà un total de 90 partits. A nivell de clubs jugà al Vaasan Sport, Tampereen Ilves, Toronto Aeros, Beatrice Aeros i DHC Lyss.

Referències

Medallistes als Jocs Olímpics d'hivern de 1998
Persones de Vaasa
Jugadores d'hoquei sobre gel finlandeses

redpajama_set_name: RedPajamaWikipedia


text:
> News & Events > News
More doors open for Toronto-based ShopLocket to power the next wave of e-commerce
OCE's SmartStart program in partnership with Extreme Startups helped position the tech start-up for $1-million in additional seed financing
As one of the successful participants in the first round of the OCE/Extreme Startups SmartStart business accelerator program, ShopLocket, has proven to the investment community it has the potential to disrupt the e-commerce space and power a new wave of merchants.
The innovative new start-up secured $1-million in a seed financing round co-led by Rho Canada Ventures and Valar Ventures, the global investment vehicle backed by Peter Thiel. ShopLocket is Thiel's first investment in a Canadian company through the fund. Other participants in the round included BDC Venture Capital, Relay Ventures, Extreme Venture Partners and a handful of private investors.
ShopLocket is turning the traditional destination commerce model on its head. The company has made selling online to fans and followers as easy as sharing a link or embedding a video. Sellers can easily share products on popular social media sites such as Facebook, Twitter, Pinterest or any existing blog/website; no storefront to setup, no need to submit their product to an ill-suited or expensive marketplace.
The start-up was born after co-founder Katherine Hague needed to sell some custom made t-shirts online. At the time, setting up a traditional e-commerce site was too costly and too time consuming. Using an existing marketplace such as eBay or Craigslist did not meet her needs and thus ShopLocket was born.
The accumulated funds will be used to grow ShopLocket's marketing and development team. The start-up recently added a director of business development and a second developer to its team and plans to add a designer and a third developer within the year.
ShopLocket participated in OCE's SmartStart Program in partnership with Extreme Startups.The program supports Ontario's next generation of Science, Technology, Engineering and Mathematic (STEM) entrepreneurs (post-secondary students and recent graduates) by facilitating entrepreneurship training and development.
OCE Media Resources
News From Our Companies
Check out how OCE-supported companies are making news.
Visit the OCE Blog
Learn insights about Ontario's innovation ecosystem and read stories about the province's leading change-makers.

redpajama_set_name: RedPajamaCommonCrawl


text:
// Generated by the protocol buffer compiler.  DO NOT EDIT!
// source: google/cloud/gaming/v1/realms.proto

package com.google.cloud.gaming.v1;

public interface ListRealmsResponseOrBuilder
    extends
    // @@protoc_insertion_point(interface_extends:google.cloud.gaming.v1.ListRealmsResponse)
    com.google.protobuf.MessageOrBuilder {

  /**
   *
   *
   *

   * The list of realms.
   *

   *
   * repeated .google.cloud.gaming.v1.Realm realms = 1;
   */
  java.util.List<com.google.cloud.gaming.v1.Realm> getRealmsList();
  /**
   *
   *
   *

   * The list of realms.
   *

   *
   * repeated .google.cloud.gaming.v1.Realm realms = 1;
   */
  com.google.cloud.gaming.v1.Realm getRealms(int index);
  /**
   *
   *
   *

   * The list of realms.
   *

   *
   * repeated .google.cloud.gaming.v1.Realm realms = 1;
   */
  int getRealmsCount();
  /**
   *
   *
   *

   * The list of realms.
   *

   *
   * repeated .google.cloud.gaming.v1.Realm realms = 1;
   */
  java.util.List<? extends com.google.cloud.gaming.v1.RealmOrBuilder> getRealmsOrBuilderList();
  /**
   *
   *
   *

   * The list of realms.
   *

   *
   * repeated .google.cloud.gaming.v1.Realm realms = 1;
   */
  com.google.cloud.gaming.v1.RealmOrBuilder getRealmsOrBuilder(int index);

  /**
   *
   *
   *

   * Token to retrieve the next page of results, or empty if there are no more
   * results in the list.
   *

   *
   * string next_page_token = 2;
   *
   * @return The nextPageToken.
   */
  java.lang.String getNextPageToken();
  /**
   *
   *
   *

   * Token to retrieve the next page of results, or empty if there are no more
   * results in the list.
   *

   *
   * string next_page_token = 2;
   *
   * @return The bytes for nextPageToken.
   */
  com.google.protobuf.ByteString getNextPageTokenBytes();

  /**
   *
   *
   *

   * List of locations that could not be reached.
   *

   *
   * repeated string unreachable = 3;
   *
   * @return A list containing the unreachable.
   */
  java.util.List<java.lang.String> getUnreachableList();
  /**
   *
   *
   *

   * List of locations that could not be reached.
   *

   *
   * repeated string unreachable = 3;
   *
   * @return The count of unreachable.
   */
  int getUnreachableCount();
  /**
   *
   *
   *

   * List of locations that could not be reached.
   *

   *
   * repeated string unreachable = 3;
   *
   * @param index The index of the element to return.
   * @return The unreachable at the given index.
   */
  java.lang.String getUnreachable(int index);
  /**
   *
   *
   *

   * List of locations that could not be reached.
   *

   *
   * repeated string unreachable = 3;
   *
   * @param index The index of the value to return.
   * @return The bytes of the unreachable at the given index.
   */
  com.google.protobuf.ByteString getUnreachableBytes(int index);
}

redpajama_set_name: RedPajamaGithub


Jerry was born November 10, 1942 in Cisco, Texas and passed away peacefully in his home on January 21, 2016. Memorial services will be held 2:30 p.m. Sunday, February 7, 2016 at Sylvester Baptist Church to celebrate the vibrant life of Jerry Glen "Papa" Carey.
He was the son of the late J.A. Carey and Minnie L. Jones Carey. Jerry was happily married to the love of his life, Nina Jean, since September 12, 1964. They enjoyed life together for 51 years.
Jerry was preceded in death by both of his parents; three brothers; one sister and also by his daughter, Lucretia June Carey Moffett.
Survivors include his wife, Nina Jean Carey of Anson; two daughters, Teresa Duncan of South Carolina, Amanda Pippin and husband Russel of Anson; a sister, June Hindman of Missouri; son in law, Shannon Moffett of McCaulley; three grandsons, Robert Pippin and wife Sabrina of Anson, Matthew Pippin and wife Leah of Lubbock, Bryce Pippin of Anson; three great grandkids, Riley, Baleigh and Berklee Pippin, all of Anson; and numerous nieces and nephews.
To send flowers or a memorial gift to the family of Jerry Glen Carey please visit our Sympathy Store.


text:
CT.GOV Information
COVID-19 Research Involving Humans
Taskforces and Committees
COVID-19 Clinical Trials
COVID-19 Non-Clinical Trials
Biospecimens Repository
Consent Form Templates
Investigator Manual
Referenced Materials
Study Conduct
General Tools and Links
Required Training
CICERO Instructional Videos
IRB Process Videos
HRP Lecture Videos
HRP Office
HRPO Personnel
Performance Informatics
For Research Participants
UMB HRP Components
HRPP Plan
SharePoint - IRB Members Site
Human Research Protections Office
hrpo@umaryland.edu
620 W. Lexington St.
Office hours are 8:30 a.m.-4:30 p.m., Monday through Friday.
* The Human Research Protections Office is working remotely and is ready to assist our community during the COVID-19 pandemic. During this time, we request you contact us via email to promote a prompt response. With apologies, currently voicemail messages may be delayed in reaching us and cause a longer wait time for us to respond to your requests. We thank you for your patience and look forward to assisting you.
Clinical Trials Registration Information
10-31-2019: Update to the ClinicalTrials.gov PRS website
This change enables the record to differentiate between studies affiliated with the University of Maryland, Baltimore and the University of Maryland, College Park.
ClinicalTrials.gov Instructions for the Research Community
ClinicalTrials.gov Checklist UMB
Clinical Trials Registration and Results Reporting Taskforce
Recorded Seminar: Ensuring Institutional Compliance with ClinicalTrials.gov - Feb 12th, 2019
For general clinicaltrials.gov questions please contact:
Jan Martinez
IRB Manager
jan.martinez@umaryland.edu
Martina Miller
IRB Analyst
martina.miller@umaryland.edu
* To Obtain a PRS account needed to create a clinicaltrials.gov entry, please contact Martina Miller and provide your name, phone number, and email address
NIH Implemented Reforms
Over the last few years, the National Institutes of Health has begun implementing a series of clinical trials reforms designed to enhance the accountability and transparency of clinical research. These reforms affect grants and contracts involving clinical trials, and several changes took effect on January 25, 2018.
Below are several key NIH reforms of importance to the NHLBI research community:
Clinical trial-specific funding opportunities: Applications and proposals involving clinical trials with due dates on or after January 25, 2018, must be submitted to a funding opportunity announcement (FOA) or request for proposal (RFP) that explicitly states it will accept clinical trials. After January 25, 2018, the NIH will only accept clinical trial applications when submitted to parent announcements or other FOAs that specify clinical trials.
New Human Subjects and Clinical Trial Information form: The NIH will require the use of a new application form that consolidates all information related to human subjects and clinical trials into one place, and also expands the information required for studies that meet the NIH definition of a clinical trial. This form will be included in the newFORMS-E Application Packages to be used for all due dates after January 25, 2018.
Single IRB policy for multi-site research: For applications with due dates after January 25, 2018, and contract solicitations published after January 25, 2018, the NIH expects that all sites participating in multi-site studies that involve non-exempt human subjects research funded by the NIH will use a single Institutional Review Board (sIRB) to conduct the ethical review required for the protection of human subjects. This requirement applies to all applicable human subjects studies, not just trials.
Registering and reporting for NIH-funded clinical trials: All NIH-funded clinical trials are expected to register and submit results information to ClinicalTrials.gov to help ensure that information about clinical trials and their results are made publicly available, in a timely manner.
Clinical Trials Protocol Template for Phase II and III Clinical Trials Conducted Under an IND or IDE: Investigators are encouraged to use a template and electronic protocol-writing tool, developed by the NIH and the Food and Drug Administration, to help evaluate the scientific basis of their assumptions, minimize uncertainty in the interpretation of outcomes, and prevent loss of data.
Good Clinical Practice Training: The NIH expects all NIH-funded clinical investigators and trial staff who are involved in the design, conduct, oversight, or management of clinical trials to be trained in Good Clinical Practice to help ensure the safety, integrity, and quality of clinical trials.
Resources for more information:
Clinical Trial Requirements for Grants and Contracts
Why Changes to Clinical Trial Policies?
Overview of New NIH Policies on Human Subjects Research (tutorial)
NHLBI Funding Opportunities and Related Resources
Any questions? Please email: NHLBIClinicalTrials@mail.nih.gov


text:
DJIBOUTI Issue dated 16/09/2016
Heightened tension between Prime Minister and Djama Speed
The minister for health and presidential couple's son-in-law Djama Elmi [...] (135 words)
DJIBOUTI 12/05/2017
Guelleh no longer trusts his prime minister
Are the days of prime minister Abdoulkader Kamil Mohamed counted? According to our sources, president Ismail Omar Guelleh wants to [...]
Expulsions of senior civil servants continue
After expelling the ministers in Ismaïl Omar Guelleh's third mandate [...]
Power vacuum on backdrop of family and tribal tension
Although Ismaïl Omar Guelleh came back from vacation early, towards the end of August, but has taken a step back [...]
Guelleh seeks protection from his own army
Djibouti's chief-of-staff, general Zakaria Cheikh Ibrahim, has become a marked man. Ali Hassan Bahdon, defense minister and son-in-law of president [...]
How the Prime Minister booted the former ministers out
With the green light from President Ismaïl Omar Guelleh (IOG), [...]
Djama Speed can't manage to keep Aroun Omar Aden by his side
Djama Elmi Okieh, known as Djama Speed, the minister for [...]
IOG prepares ground for president Djama Speed
Barely re-elected for a fourth mandate, President Ismaïl Omar Guelleh is already laying the groundwork for his successor. He is [...]
Abdoulkader Kamil Mohamed
Djama Ali Guelleh
Djama Elmi Okieh
Djama Speed
Electricite de Djibouti
Ismaïl Omar Guelleh
Mohamed Fouad Abdo
Office national de l'eau et de l'assainissement de Djibouti


text:
Q: 'The type or namespace name missing' I followed every step properly to develop an app in xamarin studio as shown in(https://developer.xamarin.com/guides/android/getting_started/hello,android/hello,android_quickstart/)
But, After building the solution there 11 errors as
1.'The type or namespace name Android could not be found.(Are you missing a using directive or an assembly reference?)'
2.'The type or namespace name Activity could not be found.(Are you missing a using directive or an assembly reference?)'
3.'The type or namespace name Bundle could not be found.(Are you missing a using directive or an assembly reference?)'
How to get rid of these errors? Please help
Here's the code---
    using System;
    using Android.App;
    using Android.Content;
    using Android.Runtime;
    using Android.Views;
    using Android.Widget;
    using Android.OS;

        namespace PhoneWord
        {
         [Activity (Label = "PhoneWord", MainLauncher = true, Icon ="@drawable/icon")]
  public class MainActivity : Activity
{
    int count = 1;

    protected override void OnCreate (Bundle bundle)
    {
        base.OnCreate (bundle);

        // Set our view from the "main" layout resource
        SetContentView (Resource.Layout.Main);

        // Get our UI controls from the loaded layout:
        EditText phoneNumberText = FindViewById<EditText>(Resource.Id.PhoneNumberText);
        Button translateButton = FindViewById<Button>(Resource.Id.TranslateButton);
        Button callButton = FindViewById<Button>(Resource.Id.CallButton);
        // Disable the "Call" button
        callButton.Enabled = false;

        // Add code to translate number
        string translatedNumber = string.Empty;

        translateButton.Click += (object sender, EventArgs e) =>
        {
            // Translate user's alphanumeric phone number to numeric
            translatedNumber = Core.PhonewordTranslator.ToNumber(phoneNumberText.Text);
            if (String.IsNullOrWhiteSpace(translatedNumber))
            {
                callButton.Text = "Call";
                callButton.Enabled = false;
            }
            else
            {
                callButton.Text = "Call " + translatedNumber;
                callButton.Enabled = true;
            }
        };

        // Get our button from the layout resource,
        // and attach an event to it
        /*Button button = FindViewById<Button> (Resource.Id.myButton);

        button.Click += delegate {
            button.Text = string.Format ("{0} clicks!", count++);
        };*/
    }
  }
}

Thank you

A: I solved the issue by adding a reference to
Mono.Android.dll

Located under :
C:\Program Files (x86)\Microsoft Visual Studio\2017\Community\Common7\IDE\ReferenceAssemblies\Microsoft\Framework\MonoAndroid\v8.1\

A: It looks like your project namespace contains the Android like in MyProject.Android. This is mostly the cause for what you describe.
Change your namespace and it should work.


text:
Nicholas Reese Art (born January 13, 1999) is an American former child actor. He is best known for his roles in the films Syriana (2005) and The Nanny Diaries (2007).

Life and career
Art was born in Milford, Connecticut on January 13, 1999, the son of Sharon and Richard Art.

He began his career at the age of three, playing Zach Spaulding on the soap opera Guiding Light (2002–2008). He then appeared in the feature films Syriana (2005) and The Nanny Diaries (2007), the television series Cashmere Mafia (2008) and the television film Taking Chance (2009). His last film job was an uncredited role in Knight and Day (2010).

Awards and nominations

References

External links

1999 births
Living people
American male child actors
American male film actors
American male soap opera actors
American male television actors
21st-century American male actors
Male actors from Connecticut
People from Milford, Connecticut


Representative Example: Finance subject to status, terms and conditions apply. Lyons Holiday Parks are committed to responsible lending. Monthly payments: £316.58, optional final repayment: £316.58, cash price: £23,600, deposit: £4,600, purchase fee (included in optional final repayment): £10, total amount of credit: £19,000, total amount repayable: £31,192.72, Interest rate (fixed): 10.11%, representative APR, 10.6%. Lyons Holiday Parks is a trading style of Lyons Holiday Park Ltd. who are acting as a credit broker and not a lender.
Available immediately is this fantastic three bedroom spacious holiday home. Offering a very comfortable large wrap around sofa in the lounge area there is plenty of room for all, even if extra guests decide to stay the night as it also converts to a pull-out double bed. Separate seated dining area that is also freshly upholstered. Gas fire and fresh new curtains. Well appointed kitchen with gas cooker and fridge, modern work tops and units. Shower room with large shower and wash basin and separate toilet with sink. Three fantastic size bedrooms with space and storage available.


text:
Welcome back to Saturday School
Herron's Saturday School, established in 1922, offers high-quality art instruction to youth and teens, as well as adults who want to learn or improve their technical art and design skills, explore new art forms, and build a portfolio.
Saturday School will begin in September 2021 and conclude in April 2022. Enrollment is divided into three groups: students in grades 1 through 4, students in grades 5 through 8, and students in grades 9 through 12. For adults 18 and up, there are two sections: one in two-dimensional studio art and one in three-dimensional studio art. Pricing varies based on the course offering.
Learn more about Herron Community Learning Program's Saturday School programming by using the accordion dropdowns. Scroll down to see the class descriptions and register, or jump to fall 2021 or spring 2022.
Dates, times, and locations
There are six Saturday School classes each semester:
Fall: Sept. 25; Oct. 2, Oct. 16, Oct. 23, Oct. 30, and Nov. 6.
Spring: Feb. 19, Feb. 26, March 5, March 26, April 2, and April 9.
The following are the class times and locations for each group:
Grades 1 through 8: Classes are held in Eskenazi Hall from 9 a.m. to 12 p.m.
Grades 9 through 12 and adults ages 18 and up: Classes are held in Eskenazi Hall and the Eskenazi Fine Arts Center from 1 to 3 p.m., with open studio hours until 5 p.m. More information can be found by scrolling down to the class descriptions.
Class fees
Grades 1 through 8: $300 per semester
Grades 9 through 12: $350 per semester
Adults ages 18 and up: $350 per semester
Scholarships are available for students in grades 1 through 12. To apply, complete our online scholarship application form.
Students in grades 4 or 8 who wish to "test out" of their current grade level and into a higher group should submit a portfolio of work to sschool@iupui.edu for consideration.
Culminating exhibitions
Herron Community Learning Programs will host a culminating exhibition of student work at Eskenazi Fine Arts Center each semester. The fall exhibition will take place on Nov. 20, 2021, at 5 p.m., and the spring exhibition will take place on April 16, 2022, at 5 p.m.
Private and group lessons
Private and group art lessons are available during the fall and spring semesters, with scheduling and pricing tailored to each student's individual needs. Please email sschool@iupui.edu if you have any questions.
Discounted rates
Saturday School programming, as well as private and group art lessons, are available at reduced rates for IU affiliates and Indiana educators. Please email sschool@iupui.edu if you have any questions.
Confirmed enrollment
Although completing the online registration form secures a student's place in a Saturday School class, enrollment is not guaranteed until full payment is received.
Art Adventurers
This spring class will take students in grades 1 through 4 on a journey into the unknown world of creativity. Participants will draw, paint, and print as they travel through outer space, encountering galaxies and planets, while designing, building, and sculpting imagined creatures, architecture, and characters inspired by diverse contemporary artists. Your intrepid art adventurer will also break down creative barriers through performance art, photography, and innovative studio practices.
Dates: Feb. 19, 26; March 5, 26; April 2, 9
Grades: 1 through 4
Location: Eskenazi Hall
Times: 9 a.m. – Noon
Cost: $300 per semester
Fantastic Storytellers
This spring, students in grades 5 through 8 will create drawings, paintings, and prints of their own imagined environments complete with original and inspired people, places, and things. Participants will also design and build three-dimensional miniature rooms using 3-D printing and interior design techniques, as well as create imagined figures and forms out of polymer clay and create performance-based videos of short stories inspired by their own daily lives.
High School Studio Art
Each Saturday, students in grades 9 through 12 can delve into the processes and techniques of drawing and illustration, painting, printmaking, designing, building, animation, performance, and documentation, all while learning about careers in the arts. Projects and instruction will differ from those of the Saturday School session in fall, but will concentrate on similar media processes. This class is appropriate for both beginning and advanced students, as all students will be encouraged to produce work that contributes to their personal art portfolios.
Grades: 9 through 12
Times: 1 – 3 p.m., with a supervised open studio until 5 p.m.
2-D Studio Art for Adults (Ages 18+)
Adults ages 18 and up can explore painting, printmaking, drawing and illustration, paper- and book-making, and photography processes and techniques in the spring. Projects and instruction will differ from those of the Saturday School session in fall, but will concentrate on similar media processes. Both beginning and advanced students are welcome to enroll and create in our studios.
Times: 1 – 3 p.m., with an open studio until 5 p.m.
Adults ages 18 and up can explore processes and techniques used in ceramics with hand-building and wheel-throwing, as well as sculpture, woodworking, and 3-D printing. Projects and instruction will differ from those of the Saturday School session in fall, but will concentrate on similar media processes. Both beginning and advanced students are welcome to enroll and create in our studios.
Location: Eskenazi Fine Arts Center
Global Explorers
Students in grades 1 through 4 can embark on a global culture expedition in Herron's studios by investigating diverse artists and mixed media approaches in the visual arts. Participants will draw, paint, and print while researching cultural and geographical differences around the world, as well as design, build, and sculpt original works of art inspired by a variety of contemporary artists. Performances, photography, and innovative studio practices will also encourage your global explorer to imagine the infinite possibilities of their creativity.
Dates: Sept. 25; Oct. 2, 16, 23, 30; Nov. 6
Time: 9 a.m. – Noon
Registration for fall 2021 Saturday School classes is now closed.
Epic Illustrators
Illustrators in grades 5 through 8 will create original adventures featuring creatures, characters, and storylines from their own imaginations in this Saturday School class. Participants will practice figure drawing, expand their color vocabulary with gouache painting techniques, and create their own storyboards using relief printmaking processes. They will investigate sequential storytelling by creating stop-motion animations, board games, and toys.
Every Saturday, students in grades 9 through 12 can participate in unique excursions into the processes and techniques of drawing and illustration, painting, printmaking, designing, building, animation, performance, and documentation, all while learning about careers in the arts. This class is appropriate for both beginning and advanced students, as all participants will be encouraged to create work for their personal art portfolios.
Time: 1 – 3 p.m., with a supervised open studio until 5 p.m.
Adults ages 18 and up can return to Eskenazi Hall on Saturdays for a semester of two-dimensional art-making. Explore processes and techniques in painting, printmaking, drawing and illustration, paper- and book-making, and photography. Both beginning and advanced students are welcome to enroll and create in our studios.
Time: 1 – 3 p.m., with an open studio until 5 p.m.
Adults ages 18 and up are invited to return to Eskenazi Fine Arts Center on Saturdays for a semester of three-dimensional art-making. Explore processes and techniques used in ceramics with hand-building and wheel-throwing, as well as sculpture, woodworking, and 3-D printing. Both beginner and advanced students are welcome to enroll and create in our studios.
Times: 1 – 3 p.m., with an open studio until 5 p.m
Financial aid and tuition discounts
Herron provides needs-based scholarships to eligible students and families. Funds are limited and granted on a first-come, first-served basis. To apply, complete our online scholarship application form.
Siblings and students of IUPUI employees receive a 10% discount. Contact sschool@iupui.edu for the discount code or questions about scholarships.


text:
Francis Finch (c. 1602August 1677) of Rushock, Wiltshire, was an English politician who sat in the House of Commons of England from 1661 to 1677.

He was known as a Protestant and supporter of the monarchy and was rewarded during the Restoration. Finch was a Member of Parliament (MP) for Winchelsea in the Cavalier Parliament from 1661 until his death in 1677.

References

English MPs 1661–1679
1600s births
1677 deaths
Year of birth uncertain


text:
<?php

namespace Brickrouge;

use ICanBoogie\Errors;

/**
 * A `<FORM>` element.
 */
class Form extends Element implements Validator
{
	/**
	 * Set to true to disable all the elements of the form.
	 */
	const DISABLED = '#form-disabled';

	/**
	 * Used to provide hidden values. Each key/value pair of the array is used to create
	 * an hidden input element with key as `name` attribute and value as `value` attribute.
	 */
	const HIDDENS = '#form-hiddens';

	/**
	 * Used by elements to define a form label, this is different from the
	 * {@link Element::LABEL}, which wraps the element in a `<LABEL>` element, the form label is
	 * associated with the element but its layout depend on the form renderer.
	 */
	const LABEL = '#form-label';

	/**
	 * Complement to the {@link LABEL} tag. Its layout depends on the form renderer.
	 */
	const LABEL_COMPLEMENT = '#form-label-complement';

	/**
	 * If true possible alert messages are not displayed.
	 */
	const NO_LOG = '#form-no-log';

	/**
	 * Values for the elements of the form. The form recursively iterates through its
	 * children to set their values, if their values it not already set (e.g. non null).
	 */
	const VALUES = '#form-values';

	/**
	 * The class name of the renderer to use to render the children of the form. If no
	 * renderer is defined, children are simply concatenated.
	 */
	const RENDERER = '#form-renderer';

	/**
	 * Defines the actions of the form.
	 *
	 * @see render_actions()
	 */
	const ACTIONS = '#form-actions';

	/**
	 * Defines form errors.
	 */
	const ERRORS = '#form-errors';

	/**
	 * Returns a unique form name.
	 *
	 * @return string
	 */
	static protected function get_auto_name()
	{
		return 'form-autoname-' . self::$auto_name_index++;
	}

	static protected $auto_name_index = 1;

	/**
	 * Hidden values, initialized with the {@link HIDDENS} tag.
	 *
	 * @var array[string]string
	 */
	public $hiddens = [];

	/**
	 * Name of the form.
	 *
	 * @var string
	 */
	public $name;

	/**
	 * Default attributes are added to those provided using a union:
	 *
	 * - `action`: If the `id` attribute is provided, `action` is set to "#<id>".
	 * - `method`: "POST"
	 * - `enctype`: "multipart/form-data"
	 * - `name`: The value of the `id` attribute or a name generated with the {@link get_auto_name()} method
	 *
	 * If `method` is different than "POST" then the `enctype` attribute is unset.
	 *
	 * @param array $attributes
	 */
	public function __construct(array $attributes=[])
	{
		$attributes += [

			'action' => isset($attributes['id']) ? '#' . $attributes['id'] : '',
			'method' => 'POST',
			'enctype' => 'multipart/form-data',
			'name' => isset($attributes['id']) ? $attributes['id'] : self::get_auto_name()

		];

		if (strtoupper($attributes['method']) != 'POST')
		{
			unset($attributes['enctype']);
		}

		$this->name = $attributes['name'] ?: self::get_auto_name();

		parent::__construct('form', $attributes);
	}

	/**
	 * Renders the object into an HTML string.
	 *
	 * Before rendering the object form elements are altered according to the {@link VALUES} and
	 * {@link DISABLED} tags and previous validation errors.
	 */
	public function __toString()
	{
		$values = $this[self::VALUES];
		$disabled = $this[self::DISABLED];

		$name = $this->name;
		$errors = null;

		if ($name)
		{
			$errors = retrieve_form_errors($name);
		}

		if ($values || $disabled || $errors)
		{
			if ($values)
			{
				$values = array_flatten($values);
			}

			if (!$errors)
			{
				$errors = new Errors();
			}

			$this->alter_elements($values, $disabled, $errors);
		}

		return parent::__toString();
	}

	/**
	 * Override the method to map the {@link HIDDENS} tag to the {@link $hiddens} property.
	 *
	 * @inheritdoc
	 */
	public function offsetSet($attribute, $value)
	{
		parent::offsetSet($attribute, $value);

		if ($attribute == self::HIDDENS)
		{
			$this->hiddens = $value;
		}
	}

	/**
	 * Returns a recursive iterator.
	 *
	 * A {@link RecursiveIterator} iterator is created to traverse the children of the form.
	 *
	 * The recursive iterator is created with the {@link SELF_FIRST} mode.
	 *
	 * @return \RecursiveIteratorIterator
	 */
	public function getIterator()
	{
		return new \RecursiveIteratorIterator(new RecursiveIterator($this), \RecursiveIteratorIterator::SELF_FIRST);
	}

	/**
	 * @var Element[] The required elements of the form.
	 */
	protected $required = [];

	/**
	 * @var string[] Booleans found in the form.
	 */
	protected $booleans = [];

	/**
	 * @var Element[] Elements of the form with a validator.
	 */
	protected $validators = [];

	/**
	 * @var callable Validator callback of the form.
	 */
	protected $validator;

	/**
	 * The method alters the {@link $required}, {@link $validators} and {@link $validator}
	 * properties required for the serialization.
	 *
	 * The following properties are exported: name, required, validators and validator.
	 *
	 * @return array
	 */
	public function __sleep()
	{
		$required = [];
		$booleans = [];
		$validators = [];

		foreach ($this as $element)
		{
			$name = $element['name'];

			if (!$name)
			{
				continue;
			}

			if ($element[Element::REQUIRED])
			{
				$required[$name] = self::select_element_label($element);
			}

			if ($element->tag_name == 'input')
			{
				if ($element['type'] == 'checkbox')
				{
					$booleans[$name] = true;
				}
			}
			else if ($element->type == Element::TYPE_CHECKBOX_GROUP)
			{
				foreach ($element[self::OPTIONS] as $option_name => $dummy)
				{
					$booleans[$name . '[' . $option_name . ']'] = true;
				}
			}

			if ($element[self::VALIDATOR] || $element[self::VALIDATOR_OPTIONS] || $element instanceof Validator)
			{
				$validators[$name] = $element;
			}
		}

		$this->required = $required;
		$this->booleans = $booleans;
		$this->validators = $validators;
		$this->validator = $this[self::VALIDATOR];

		#
		# we return the variables to serialize, we only export variables needed for later
		# validation.
		#

		return [ 'name', 'required', 'booleans', 'validators', 'validator' ];
	}

	/**
	 * If a rendered is defined it is used to render the children.
	 *
	 * The rendered is defined using the {@link RENDERER} attribute.
	 *
	 * @inheritdoc
	 */
	protected function render_children(array $children)
	{
		/* @var $renderer callable*/

		$renderer = $this[self::RENDERER];

		if ($renderer)
		{
			if (is_string($renderer))
			{
				$class = $renderer;

				if (!class_exists($class))
				{
					$class = 'Brickrouge\Renderer\\' . $class;
				}

				$renderer = new $class();
			}

			return $renderer($this);
		}

		return parent::render_children($children);
	}

	/**
	 * Add hidden input elements and log messages to the inner HTML of the element
	 * being converted to a string.
	 *
	 * @inheritdoc
	 */
	protected function render_inner_html()
	{
		$inner_html = parent::render_inner_html();
		$hiddens = $this->render_hiddens($this->hiddens);

		if (!$inner_html)
		{
			$this->add_class('has-no-content');
		}
		else
		{
			$this->remove_class('has-no-content');
		}

		#
		# alert message
		#

		$alert = null;

		if (!$this[self::NO_LOG])
		{
			$name = $this->name;
			$errors = retrieve_form_errors($name);

			if ($errors)
			{
				$alert = $this->render_errors($errors);

				store_form_errors($name, []); // reset form errors.
			}
		}

		#
		# actions
		#

		$actions = $this[self::ACTIONS];

		if ($actions)
		{
			$this->add_class('has-actions');

			$actions = $this->render_actions($actions);
		}
		else
		{
			$this->remove_class('has-actions');
		}

		if (!$inner_html && !$actions)
		{
			throw new ElementIsEmpty();
		}

		return $hiddens . $alert . $inner_html . $actions;
	}

	/**
	 * Renders errors as an HTML element.
	 *
	 * An {@link Alert} object is used to render the provided errors.
	 *
	 * @param string|\ICanBoogie\Errors $errors.
	 *
	 * @return string
	 */
	protected function render_errors($errors)
	{
		return (string) new Alert($errors);
	}

	/**
	 * Renders actions using an {@link Actions} element.
	 *
	 * @param mixed $actions
	 *
	 * @return string Return the actions block.
	 */
	protected function render_actions($actions)
	{
		return (string) new Actions($actions, [ 'class' => 'form-actions' ]);
	}

	/**
	 * Renders hidden values.
	 *
	 * @param array $hiddens
	 *
	 * @return string
	 */
	protected function render_hiddens(array $hiddens)
	{
		$html = '';

		foreach ($hiddens as $name => $value)
		{
			#
			# we skip undefined values
			#

			if ($value === null)
			{
				continue;
			}

			$html .= '<input type="hidden" name="' . escape($name) . '" value="' . escape($value) . '" />';
		}

		return $html;
	}

	/**
	 * Alters the elements according to the state of the form.
	 *
	 * @param array $values
	 * @param bool $disabled true if the form is disabled, false otherwise.
	 * @param array|\ICanBoogie\Errors $errors The validation errors.
	 */
	protected function alter_elements($values, $disabled, $errors)
	{
		foreach ($this as $element)
		{
			#
			# disable the element if the form is disabled.
			#

			if ($disabled)
			{
				$element['disabled'] = true;
			}

			$name = $element['name'];

			if (!$name)
			{
				continue;
			}

			#
			# if the element is referenced in the errors, we set its state to 'error'
			#

			if (isset($errors[$name]))
			{
				$element[Element::STATE] = 'error';
			}

			#
			# set value
			#

			if ($values && array_key_exists($name, $values))
			{
				$type = $element['type'];
				$value = $values[$name];

				#
				# we don't override the `value` or `checked` attributes if they are already defined
				#

				if ($type == 'checkbox')
				{
					if ($element['checked'] === null)
					{
						$element['checked'] = !empty($value);
					}
				}
				else if ($type == 'radio')
				{
					if ($element['checked'] === null)
					{
						$element_value = $element['value'];
						$element['checked'] = $element_value == $value;
					}
				}
				else if ($element['value'] === null)
				{
					$element['value'] = $value;
				}
			}
		}
	}

	/*
	 * Save and restore.
	 */

	const STORED_KEY_NAME = '_brickrouge_form_key';

	/**
	 * Save the form in the session for future validation.
	 *
	 * @return string The key used to identify the form save in the session.
	 */
	public function save()
	{
		$key = store_form($this);

		$this->hiddens[self::STORED_KEY_NAME] = $key;

		return $this;
	}

	/**
	 * Loads a form previously saved in session.
	 *
	 * @param string $key The key used to identify the form to load, or an array in which
	 * {@link STORED_KEY_NAME} defines the key.
	 *
	 * @return Form
	 *
	 * @throws \Exception if the form cannot be retrieved.
	 */
	static public function load($key)
	{
		if (is_array($key))
		{
			if (empty($key[self::STORED_KEY_NAME]))
			{
				throw new \Exception('The key to retrieve the form is missing.');
			}

			$key = $key[self::STORED_KEY_NAME];
		}

		$form = retrieve_form($key);

		if (!$form)
		{
			throw new \Exception('The form has expired.');
		}

		$form[self::VALIDATOR] = $form->validator;

		return $form;
	}

	/**
	 * Checks if a previously saved form exists for a given key.
	 *
	 * @param string $key The key used to identify the form.
	 *
	 * @return boolean Return `true` if the form exists.
	 */
	static public function exists($key)
	{
		check_session();

		return !empty($_SESSION['brickrouge.saved_forms'][$key]);
	}

	static public function select_element_label($element)
	{
		$label = $element[self::LABEL_MISSING];

		if (!$label)
		{
			$label = $element[Form::LABEL];
		}

		if (!$label)
		{
			$label = $element[Element::LABEL];
		}

		if (!$label)
		{
			$label = $element[self::LEGEND] ?: $label;
		}

		#
		# remove HTML markups from the label
		#

		$label = t($label, [], [ 'scope' => 'element.label' ]);
		$label = strip_tags($label);

		return $label;
	}

	/**
	 * Validates the form using the provided values.
	 *
	 * @inheritdoc
	 */
	public function validate($values, Errors $errors)
	{
		#
		# validation without prior save
		#

		if (empty($values[self::STORED_KEY_NAME]))
		{
			$this->__sleep();
		}

		#
		# we flatten the array so that we can easily get values
		# for keys such as `cars[1][color]`
		#

		$values = array_flatten($values);

		$this->values = $values;

		#
		# process required values
		#

		$validators = $this->validators;

		foreach ($validators as $identifier => $element)
		{
			$element->form = $this;
			$element->name = $identifier;
			$element->label = self::select_element_label($element);
		}

		#
		# process required elements
		#

		$this->validate_required_elements($this->required, $validators, $values, $errors);

		#
		# process elements validators
		#
		# note: If the value for the element is `null` and the value is not required the element's
		# validator is *not* called.
		#

		foreach ($validators as $name => $element)
		{
			$value = isset($values[$name]) ? $values[$name] : null;

			if (($value === null || $value === '') && empty($this->required[$name]))
			{
				continue;
			}

			$element->validate($value, $errors);
		}

		// FIXME-20111013: ICanBoogie won't save the errors in the session, so we have to do it ourselves for now.

		store_form_errors($this->name, $errors);

		if (count($errors))
		{
			return false;
		}

		return parent::validate($values, $errors);
	}

	protected function validate_required_elements(array $required, array &$validators, array $values, Errors $errors)
	{
		$missing = [];

		foreach ($required as $name => $label)
		{
			if (!isset($values[$name]) || (isset($values[$name]) && is_string($values[$name]) && !strlen(trim($values[$name]))))
			{
				$missing[$name] = $this->t($label);

				#
				# The value for this required element is missing.
				# In order to avoid troubles, the element is removed
				# for the validators array.
				#

				unset($validators[$name]);
			}
		}

		if ($missing)
		{
			if (count($missing) == 1)
			{
				$errors[key($missing)] = $this->t('The field %field is required!', [ '%field' => $this->t(current($missing)) ]);
			}
			else
			{
				foreach ($missing as $name => $label)
				{
					$errors[$name] = true;
				}

				$last = array_pop($missing);

				$errors[] = $this->t('The fields %list and %last are required!', [ '%list' => implode(', ', $missing), '%last' => $last ]);
			}
		}
	}

	/*
	 * Validators
	 */

	static public function validate_email(Errors $errors, $element, $value)
	{
		if (filter_var($value, FILTER_VALIDATE_EMAIL))
		{
			return true;
		}

		$errors[$element->name] = t('Invalid email address %value for the %label element.', [ 'value' => $value, 'label' => $element->label ]);

		return false;
	}

	static public function validate_url(Errors $errors, $element, $value)
	{
		if (filter_var($value, FILTER_VALIDATE_URL))
		{
			return true;
		}

		$errors[$element->name] = t('Invalid URL %value for the %label element.', [ 'value' => $value, 'label' => $element->label ]);

		return false;
	}

	static public function validate_string(Errors $errors, $element, $value, $rules)
	{
		$messages = [];
		$args = [];

		foreach ($rules as $rule => $params)
		{
			switch ($rule)
			{
				case 'length-min':
				{
					if (strlen($value) < $params)
					{
						$messages[] = t('The string %string is too short (minimum size is :size characters)', [

							'%string' => $value,
							':size' => $params

						]);
					}
				}
				break;

				case 'length-max':
				{
					if (strlen($value) > $params)
					{
						$messages[] = t('The string %string is too long (maximum size is :size characters)', [

							'%string' => shorten($value, 32, 1),
							':size' => $params

						]);
					}
				}
				break;

				case 'regex':
				{
					if (!preg_match($params, $value))
					{
						$messages[] = t('Invalid format of value %value', [ '%value' => $value ]);
					}
				}
				break;
			}
		}

		if ($messages)
		{
			$message = implode('. ', $messages);

			$message .= t(' for the %label input element.', [ '%label' => $element->label ]);

			$errors[$element->name] = t($message, $args);
		}

		return empty($messages);
	}

	static public function validate_range(Errors $errors, $element, $value, $rules)
	{
		list($min, $max) = $rules;

		$rc = ($value >= $min && $value <= $max);

		if (!$rc)
		{
			$errors[$element->name] = t('@wdform.errors.range', [

				'%label' => $element->label,
				':min' => $min,
				':max' => $max

			]);
		}

		return $rc;
	}
}


text:
Mazar steht für:
 Mazar (Fluss), Fluss in Ecuador;
 Masar-e Scharif (Mazar i Scharif), Stadt in Afghanistan;
 Mazar (Taxkorgan), die Staatliche Schafzuchtfarm Mazar (麻扎尔种羊场) im Autonomen Kreis Taschkorgan, Uigurisches Autonomes Gebiet Xinjiang der Volksrepublik China;

und für die Personen:
 Amichai Mazar (* 1942), israelischer Archäologe
 Benjamin Mazar, eigentlich B. Maisler (1906–1995), israelischer Archäologe;
 Debi Mazar, US-amerikanische Schauspielerin;
 Eilat Mazar (1956–2021), israelische Archäologin, Enkelin von Benjamin Mazar.

Siehe auch:
 Mazar-e-Quaid


With a stay at Batavia Apartment in Perth, you'll be a 3-minute drive from nib Stadium and 5 minutes from Elizabeth Quay. This 4.5-star apartment is 4 mi (6.5 km) from Crown Perth and 5 mi (8 km) from Kings Park and Botanic Garden.
Make yourself at home in one of the 21 air-conditioned rooms featuring kitchens with full-sized refrigerators/freezers and stovetops. Rooms have private furnished balconies or patios. Complimentary wireless Internet access keeps you connected, and flat-screen televisions are provided for your entertainment. Conveniences include desks and microwaves, and housekeeping is provided on a limited basis.
Enjoy recreation amenities such as an outdoor pool or take in the view from a garden. Additional amenities at this apartment include complimentary wireless Internet access, tour/ticket assistance, and a picnic area.